The advent of recombinant DNA technology has enabled the production of various naturally occurring and synthetic proteins in organisms such as bacteria, fungi, yeast and mammalian cells. In general it involves the insertion of genes that encode a desired protein into a host organism, and utilizing the host's cellular machinery to express the gene.
Recombinant DNA technology is continually developing to achieve production of proteins in commercially acceptable yields. A limiting factor in recombinant production of proteins is the rate at which the gene encoding the desired protein is expressed. In particular, it has been found that the promoter region of a gene is critical in the transcription process of gene expression. An efficient promoter such as the trp promoter found in E. coli, binds tightly to DNA-directed RNA polymerase to initiate transcription of the gene in generating mRNA. A less efficient promoter such as the lac promoter binds RNA polymerase less tightly, resulting in a lower rate of mRNA generation.
The trp promoter has been widely used in the production of heterologous proteins because of its ability to initiate transcription. Despite its efficiency, an inherent shortcoming of the trp promoter is that it is not easily controlled. Specifically, the trp promoter is not fully repressible, i.e. it can drive transcription before the host is grown in culture to a phase appropriate for protein production. Another widely used promoter is lac which is less efficient than trp, however is more controllable.
To develop more efficient promoters, functional components of different promoters have been combined, for instance those described in U.S. Pat. No. 5,362,646. In one example, portions of the phage T7 promoter A.sub.1 (P.sub.A1) were combined with two lac operators. Specifically, the spacer region between the so called -35 and -10 regions of the T7 promoter was replaced with a modified lac operator sequence, and to control the resulting promoter hybrid, a second lac operator was introduced downstream. The resulting promoter/operator system which is incorporated on the commercially available pUHE plasmids was found to initiate transcription efficiently upon induction and yet is highly repressed before induction.
Another promoter described by Tsung et al (Proc. Natl. Acad. Sci. USA, 1990, 87:5940) comprises the efficient trp -35 region, the -10 region from the highly efficient lppP-5 promoter (a variant of lpp promoter) and a spacer derived from the lac promoter. This promoter was shown to be so highly efficient in initiating transcription as to result in cell lethality.
While various promoters have allowed improved yields of proteins in microbial hosts, there still remains a need for promoters that drive production of commercially valued proteins more efficiently.